The long-term goal of this research program is to elucidate the molecular mechanisms of transducin (Gt) signaling in rods and cones. A remarkable level of understanding has been achieved in the field on the mechanisms of transducin in the phototransduction cascade. The current focus of our research is on the mechanisms of transducin trafficking and transducin-dependent modulation of photoreceptor responses beyond phototransduction. Very little is known about transport of transducin from the inner segment (IS) to the outer segment (OS). Uncoordinated 119 protein (UNC119) has emerged as a major factor facilitating the IS->OS transport of transducin-? (G?t). We propose to investigate the structure and properties of the trafficking complex between G?t and UNC119 by Small Angle X-ray Scattering (SAXS) and X- ray crystallography. Based on the preliminary SAXS analysis, we hypothesize that a novel interface between UNC119 and G?t is essential to the UNC119-induced dissociation of Gt subunits and protein solubilization from the membrane. The interface and the proposed model of Gt solubilization by UNC119 will be probed with mutational analysis. Solubilization of Gt from IS membranes with the formation of the G?t-UNC119 complex is the initial step in the IS->OS transport of G?t, which terminates with the cargo release into the OS. A small ARF-like 3 GTPase, ARL3, will be investigated as a putative release factor in G?t trafficking. We hypothesize that G?t is released from the complex with UNC119 by a mechanism involving ARL3-induced conformational changes both within and outside the acyl-binding cavity of UNC119 and will test this mechanism. Defects in protein transport are a common cause of photoreceptor degeneration. Elucidation of molecular details of transducin trafficking will significantly contribute to the understanding of transport of peripheral membrane proteins in photoreceptors and will have important implications for retinal diseases. Finally, we will investigate a novel concept on the role of light-dispersed transducin in modulation of the synaptic output of rods. We hypothesize that G?t regulates rod-to-rod bipolar cell signaling by altering UNC119 interactions with major synaptic players, CaBP4 and RIBEYE, and/or via direct effects on the voltage-gated Ca2+ channels and synaptic machinery. The proposed analysis of the synaptic modulation by transducin may profoundly impact the established paradigm on transducin signaling in photoreceptors.

Public Health Relevance

Transducin is the key molecule mediating signal transfer in the vertebrate phototransduction cascade. The goals of the proposed research are to investigate transport mechanisms of transducin in photoreceptors though analysis of its trafficking complex with UNC119 and to elucidate transducin functions beyond phototransduction, such as its role in regulation of synaptic output of rods. These studies are directly relevant to understanding the molecular basis of human retinal diseases, because defective protein transport and dysregulation of the synaptic signaling of photoreceptor cells are a common cause of visual disorders.